Volume 5, Issue 12, Pages 1200-1207 (December 2016) Impaired histone deacetylases 5 and 6 expression mimics the effects of obesity and hypoxia on adipocyte function  Julien Bricambert, Dimitri Favre, Saška Brajkovic, Amélie Bonnefond, Raphael Boutry, Roberto Salvi, Valérie Plaisance, Mohamed Chikri, Giulia Chinetti-Gbaguidi, Bart Staels, Vittorio Giusti, Robert Caiazzo, François Pattou, Gérard Waeber, Philippe Froguel, Amar Abderrahmani  Molecular Metabolism  Volume 5, Issue 12, Pages 1200-1207 (December 2016) DOI: 10.1016/j.molmet.2016.09.011 Copyright © 2016 The Author(s) Terms and Conditions

Figure 1 Measurement of Hdac/HDAC activity and expression from adipose tissues, adipocytes and svf in diet-induced obese mice, non-obese and obese individuals. A) Hdac activity in WAT of mice fed with regular or HFD. Total proteins were prepared from WAT of mice that were fed with regular (open bars, Chow, n = 10 mice) or HFD (filled bars, HFD, n = 10 mice). HDACs activity was measured by direct colorimetric assay kit (Epigentek). Data are the mean ± SEM of 3 independent experiments (*p < 0.05). Quantification of B) Class I Hdac1/2/3/8, C) Class IIa Hdac4/5/7/9 D) Class IIb Hdac6/10 IIb and IV Hdac11 mRNA levels. The mRNA of E) Hdac5, F) Hdac6, and G) Hdac9 was quantified by PCR in adipocytes and stroma vascular fraction (SVF) that were collected from WAT from control (open bars, Chow) and obese mice (filled bars, HFD). The Hdac mRNA levels were determined by quantitative real-time PCR and were normalized against the housekeeping acidic ribosomal phosphoprotein P0 gene (Rplp0). Similar results were obtained while normalizing against TBP. The results were expressed as the fold changes over the controls. Data are the mean of ±SEM of 3 independent experiments (***P < 0.001; **P < 0.01; *P < 0.05). H) HDAC activity in VAT of non-obese and obese individuals. Total protein concentrations were prepared from VAT of non-obese (open bars, NO) or obese individuals (filled bars, Obese) and were subjected to colorimetric assay kit (Epigentek). Data are the mean ± SEM of 3 independent experiments (**P < 0.01). Quantification of I) HDAC5, J) HDAC6, and K) HDAC9 mRNA in adipocytes and SVF from VAT of non-obese (open bars, NO) or obese individuals (filled bars, Obese). The HDAC mRNA levels were determined by quantitative real-time PCR. The mRNA levels were normalized against the RPLP0 and were expressed as the fold changes over the controls. Data are the mean of ±SEM of 3 independent experiments (**P < 0.01; *P < 0.05). Molecular Metabolism 2016 5, 1200-1207DOI: (10.1016/j.molmet.2016.09.011) Copyright © 2016 The Author(s) Terms and Conditions

Figure 2 Effects of hypoxia on Hdac activity and gene expression. Measurement of A) Hdac5 and Hdac6, B) Hdac activity, C) Icer and D) Atf3 mRNA levels in hypoxic 3T3-L1 adipocyte cells. 3T3-L1 adipocyte cells were exposed for 12 h to normoxia or hypoxia (1% O2). For inducing the expression of Icer and Atf3 in C) and D), cells under normoxia or hypoxia were further incubated with DMSO (Ctl) or a mixture of cAMP-raising agents IBMX (100 μM)/Forskolin (10 μM) for 4 h. Quantification of adipokine mRNA by qRT-PCR in E) 3T3-L1 adipocyte cells that were exposed for 12 h to normoxia or hypoxia (1% O2) and F) upon silencing of Icer. 3T3-L1 adipocytes cells were electroporated with siGFP (siCtl, open bars) or siIcer (filled bars) for 72 h. The mRNA levels were normalized against the Rplp0 and were expressed as the fold changes over the controls. Data are the mean of ±SEM of 3 independent experiments (***P < 0.001; **P < 0.01; *P < 0.05). Molecular Metabolism 2016 5, 1200-1207DOI: (10.1016/j.molmet.2016.09.011) Copyright © 2016 The Author(s) Terms and Conditions

Figure 3 Effect of silencing of Hdac5 and Hdac6 levels in 3T3-L1 adipocytes. A) Efficiency of siRNAs on Hdac5 and Hdac6 content by Western Blotting. 3T3-L1 adipocytes were electroporated with 5 nmol of control small interfering RNA duplexes directed against GFP (siCtl) or with siRNAs against Hdac5 (siH5) or Hdac6 (siH6). Total proteins were subjected to Western blotting experiments for the quantification of Hdac5, Hdac6, and α-tubulin as a loading control. The figure shows the results of a representative experiment out of three. Effect of siH5 and siH6 on the B) expression of Icer, C) Atf3, and D) Glut4 mRNA. Gene expression was monitored by quantitative real-time PCR 72 h after transfection with either control small interfering RNA duplexes directed against GFP (siCtl), or siH5, or siH6. mRNA levels were normalized against the Rplp0 and were expressed as the fold changes over the controls. Data are the mean of ±SEM of at least 3 independent experiments (***P < 0.001). E) Efficiency of siH5 and siH6 on the 2-deoxy-glucose (DOG) uptake. Measurement of the labeled DOG in 3T3-L1 adipocytes cells was done 96 h after electroporation with siRNAs by measuring [1-3H]2-DOG (100 μm, 0.5 μCi/ml) uptake from unstimulated cells (open bar) or stimulated cell with 10 nM insulin (filled bar). Data are the mean of ±SEM of 4 independent experiments (**P < 0.01; *P < 0.05). Molecular Metabolism 2016 5, 1200-1207DOI: (10.1016/j.molmet.2016.09.011) Copyright © 2016 The Author(s) Terms and Conditions

Figure 4 Schematic representation for the role of Hdac5 and Hdac6 in coupling hypoxia and obesity to defective Icer expression and adipocyte dysfunction. Molecular Metabolism 2016 5, 1200-1207DOI: (10.1016/j.molmet.2016.09.011) Copyright © 2016 The Author(s) Terms and Conditions